KDO aldolase

ABSTRACT

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) and KDO aldolase (EC 4.1.2.23) isolated therefrom are disclosed. The KDO aldolase is further disclosed to have a broad substrate specificity with respect to its reverse reaction, i.e. the condensation of aldoses with pyruvate to form a wide range of 2-keto-3-deoxy-onic acids, including 2-keto-3-deoxy-nonulosonic acid, 2-keto-3-deoxy-octulosonic acid, 2-keto-3-deoxy-heptulosonic acid, and 2-keto-3-deoxy-hexulosonic acid. In particular, 3-deoxy-D-manno-2-octulosonic acid (D-KDO), a vital component of lipopolysaccharides found in the bacterial outer membrane may be synthesized from D-arabinose and pyruvate in 67% yield.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. GM 44154awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of application Ser. No. 07/933,140, filed Dec. 18,1992, now U.S. Pat. No. 5,358,859.

TECHNICAL FIELD

The invention relates to KDO aldolase (EC 4.1.2.23) having a broadsubstrate specificity with respect to its reverse reaction and tocondensation reactions employing such KDO aldolase for synthesizing abroad range of 6-9 carbon 2-keto-3-deoxy-onic acids, viz.2-keto-3-deoxy-hexulosonate, 2-keto-3-deoxy-heptulosonate,2-keto-3-deoxy-octulosonate, and 2-keto-3-deoxy-nonulosonate. Moreparticularly, the invention relates to Aureobacterium barkerei strainKDO-37-2 (ATCC 49977), to KDO aldolase produced by and isolated fromsuch bacteria, to the employment of such KDO aldose with respect to thesynthesis of 2-keto-3-deoxy-onic acids such as3-deoxy-D-manno-2-octulosonic acid (D-KDO) and to the use of protectedforms of such 2-keto-3-deoxy-onic acids for the production of 7 and 8carbon aldoses by means of radical mediated decarboxylation.

BACKGROUND OF THE INVENTION

2-Keto-3-deoxy-octulosonic acid (KDO) appears as a ketosidic componentof all Gram-negative bacteria for which a KDO determination has beenmade. More particularly, 3-deoxy-D-manno-2-octulosonic acid (D-KDO) iswidely found in Gram-negative bacteria. KDO is incorporated intolipopolysaccharides and is localized, as such, within the outer membranecompartment of Gram-negative bacteria. KDO appears to be a vitalcomponent of Gram-negative bacteria. KDO can also occur as an acidicexopolysaccharide. In such instances, the KDO can serve as part of aK-antigen.

As illustrated in FIG. 7, the biosynthetic incorporation of KDO intolipopolysaccharides consists of two steps, i.e.:

1. Activation of KDO to form CMP-KDO by means of CMP-KDO synthetase (EC2.7.7.38); and then

2. Coupling of the activated CMP-KDO to lipid A precursor to form lipidA-KDO by means of KDO transferase.

The rate-limiting step with respect to the biosynthesis of KDOcontaining lipopolysaccharides is the activation of the KDO moiety,i.e., the formation of CMP-KDO. Accordingly, inhibitors of CMP-KDOsynthetase are potentially useful as antibacterial agents.

Several chemical and enzymatic synthetic routes have been developed forthe synthesis of KDO and its analogs. One route for the chemicalsynthesis of KDO employs Cornforth's method. (Ghalambor, M. et al. J.Biol. Chem. 1966, 241, 3207 and Hershberger, C. et al. J. Biol. Chem.1968, 243, 1585.) The chemical synthesis of KDO produces multipleenantiomers. In order to obtain enantiomerically pure D-KDO, aseparation step must be incorporated into the chemical synthetic route.

Synthetic routes employing enzymes are more stereospecific than chemicalsynthetic routes. An enzymatic synthetic route employing KDO-8 phosphatesynthase and KDO-8-P phosphatase as catalysts and arabinose-5-P and PEPas substrates is illustrated in FIG. 7. (Bednarski, M. et al.Tetrahedron Letters 1988, 29, 427.) An alternative enzymatic syntheticroute employs sialic acid aldolase. (Aug e, C. et al. Tetrahedron 1990,46, 201.)

An enzymatic synthetic route employing the reverse reaction of KDOaldolase for a micromolar scale synthesis of KDO is disclosed byGhalambor. (Ghalambor, M. et al., J. Biol. Chem. 1966, 241, 3222.) Thesynthetic route described by Ghalambor employs KDO aldolase isolatedfrom Aerobacter colacae. The reverse reaction of KDO aldolase is drivenby employing high substrate levels, i.e. high concentrations ofD-arabinose and D-pyruvate. Ghalambor discloses that there is a 41%yield with this enzyme and narrow substrate specificity.

KDO aldolase (EC 4.1.2.23) is known to be inductively produced byseveral bacteria, viz. Escherichia coli, strains 0111, B, and K-12,Salmonella typhimurium, Salmonella aldelaide, and Aerobacter colacae.Ghalambor discloses that all of these known KDO aldolases havecomparable activities. For example, all of these KDO aldolases hydrolyze3-deoxy-D-manno-2-octulosonic acid to form D-arabinose and pyruvate in aforward reaction. As indicated above, Ghalambor also discloses thatknown KDO aldolase may be employed in a reverse reaction to condenseD-arabinose and pyruvate to form 3-deoxy-D-manno-2-octulosonic acid. Thesubstrate specificity of known KDO aldolases with respect to thisreverse reaction is confined to D-arabinose and has been specificallyshown to lack a measurable specificity for D-ribose in connection withthis reverse reaction.

What was needed was an enzymic synthetic route for the production of awide range of 2-keto-3-deoxy-onic acids and analogs thereof potentiallyhaving activity as inhibitors of CMP-KDO synthetase.

What was also needed was a method of converting 2-keto-3-deoxy-onicacids to high-carbon 2-deoxy aldoses.

SUMMARY OF THE INVENTION

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) and KDO aldolase(EC 4.1.2.23) isolated therefrom are disclosed therein. KDO aldolasecatalyzed condensation employing this enzyme has been demonstrated to beeffective for the synthesis of KDO and analogs. The reactions arestereospecific with formation of a new R-stereocenter at C-3 fromD-arabinose and related substrates. Decarboxylation of the aldolaseproducts provides a new route to heptose and octose derivatives.

Unlike known KDO aldolases which have a narrow substrate specificity,the KDO aldolase isolated from this source is disclosed to have a verywide substrate specificity with respect to catalyzing its reversereaction, i.e. the condensation of aldoses with pyruvate. In particular,3-deoxy-D-manno-2-octulosonic acid (D-KDO) can be synthesized fromD-arabinose and pyruvate in 67% yield. Furthermore, studies with respectto the substrate specificity of the enzyme using more than 20 naturaland unnatural sugars indicate that this enzyme widely accepts trioses,tetroses, pentoses and hexoses as substrates, especially the ones with Rconfiguration at 3position. The substituent of 2 position has littleeffect on the aldol reaction. Nine of these substrates are submitted tothe aldol reaction to prepare various 2-keto-3-deoxy-onic acids,including D-KDO, 3-deoxy-D-arabino-2-heptulosonic acid (D-DAH),2-keto-3-deoxy-L-gluconic acid (L-KDG), and3-deoxy-L-glycero-L-galacto-nonulosonic acid (L-KDN). The attack ofpyruvate appears to take place on the re face of the carbonyl group ofacceptor substrates, a facial selection complementary to sialic acidaldolase (si face attack) reactions. The aldolase products can beconverted to aldoses via radical-mediated decarboxylation. For example,decarboxylation of pentaacetyl KDO and hexaacetyl neuraminic acid givespenta-O-acetyl-2-deoxy-β,3-D-manno-heptose andpenta-O-acetyl-4-acetamido-2,4-dideoxy-β-D-glycero-D-gala cto-octose,respectively.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates saccharides having good specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 2 illustrates saccharides having fair specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 3 illustrates saccharides having poor specificity for KDO aldolaseisolated from Aureobacterium barkerei KDO-37-2.

FIG. 4 illustrates the stereochemistry of the aldol condensationcatalyzed by KDO aldolase isolated from Aureobacterium barkereiKDO-37-2.

FIG. 5 illustrates the ¹ H NMR spectrum of 6, the product from2-deoxy-D-ribose (400 MHz), CDCl₃.

FIG. 6 illustrates the chemical assignment of the ¹ H NMR spectrumcompound 6 as shown in FIG. 6.

FIG. 7 illustrates a prior art biosynthetic incorporation of KDO intolipopolysaccharides and a prior art enzymatic synthetic route employingKDO-8 phosphate synthase and KDO-8-P phosphatase as catalysts andarabinose-5-P and PEP as substrates.

FIGS. 8A-I illustrate a synthetic scheme employing an aldolasecondensation reaction and an excess of pyruvate for producing KDO from avariety of starting sugars.

FIG. 8A illustrates a synthetic scheme employing KDO aldolase andD-arabinose.

FIG. 8B illustrates a synthetic scheme employing KDO aldolase andD-ribose.

FIG. 8C illustrates a synthetic scheme employing KDO aldolase and2-deoxy-D-ribose.

FIG. 8D illustrates a synthetic scheme employing KDO aldolase andD-erythrose.

FIG. 8E illustrates a synthetic scheme employing KDO aldolase andD-glyceraldehyde.

FIG. 8F illustrates a synthetic scheme employing KDO aldolase andD-threose.

FIG. 8G illustrates a synthetic scheme employing KDO aldolase andL-glyceraldehyde.

FIG. 8H illustrates a synthetic scheme employing KDO aldolase andL-mannose.

FIG. 8I illustrates a synthetic scheme employing sialic acid aldolaseand D-mannose.

FIGS. 9A and B illustrate a synthetic route employing a decarboxylationof KDO aldolase condensation products. Additionally, FIG. 9A illustratesthe stabilization of a planar conformer of the radical intermediate,stabilized both by the electron-donating and withdrawing effects,thereby allowing the maximum interaction between the one-electron porbital and the lone pair electrons on the adjacent ring oxygen.

DETAILED DESCRIPTION

A New Source of KDO Aldolase

Aureobacterium barkerei strain KDO-37-2 (ATCC 49977) was isolated fromgarden soil using KDO as a major carbon source. The microorganism(strain KDO-37-2) grows well on LB medium. It is aerobic, gram-positive,not motile and with colonies 1-3 millimeters in diameter on LB agarplates. The colony morphology is circular, low convex, entire edge andproduces yellow pigment. Optimum growth temperature is about 30° C.Major fatty acids are anteiso-C_(15:0) and anteiso-C_(17:0). The strainwas identified as Aureobacterium barkerei according to Bergey's manual.A deposit of Aureobacterium barkerei strain KDO-37-2 was made Jul. 30,1992 with the American Type Culture Collection (ATCC) 12301 ParklawnDrive, Rockville, Md., USA 20852 and was given Accession Number ATCC49977.

The deposit with accession Number ATCC 49977 was made in compliance withthe Budapest Treaty requirements that the duration of the depositsshould be for 30 years from the date of deposit at the depository or forthe enforceable life of a U.S. patent that matures from thisapplication, whichever is longer. The cell line will be replenishedshould it become non-viable at the depository.

A preferred medium for KDO aldolase production is defined as follows:NH₄ Cl (5 grams), K₂ SO₄ (1 gram), MgSO₄ •7H₂ O (200 milligrams), CaCl₂(20 milligrams), FeSO₄ •7H₂ O (1 milligram), yeast extract (1 gram), Na₂HPO₄ •7H₂ O and KH₂ PO₄ (3 grams) in distilled water (1 liter) at pH7.2. A seed culture may be made by admixing in a 100 milliliterErlenmeyer flask 50 milliliters of the above medium together with 25microliters of a 40% glucose solution and 100 milligrams (0.2%) of KDO.The seed culture is then inoculated with a loopful of Aureobacteriumbarkerei strain KDO-37-2 (ATCC 49977). The flask is then shaken at 250r.p.m. on a gyrorotory shaker at 30° C. for 16 hours. The seed culturethus obtained may then be poured into the 1950 milliliters of the samemedium containing LDO as a major carbon source. The culture wasincubated for 24 hours at 30° C. with shaking. The cells may beharvested as a source of KDO aldolase enzyme.

For routine culture preservation, the culture can grow on LB medium andcan be incubated overnight at 30° C. This strain of Aureobacteriumbarkerei is shown to be a source of KDO aldolase (EC 4.1.2.23) having abroad substrate specificity with respect to the reverse aldolcondensation reaction.

A New Source of KDO Aldolase

KDO aldolase (EC 4.1.2.23) was first reported by Ghalambor and Heath in1966 as the enzyme responsible for the KDO degradation (FIG. 7). Aftertheir preliminary investigation on the substrate specificity as well asthe μmol scale synthesis of KDO, no synthetic application of this enzymehas been reported, while the related enzyme N-acetylneuraminic acid(sialic acid) aldolase has been extensively studied.

It is disclosed herein that the Gram-positive bacterium Aureobacteriumbarkerei strain KDO-37-2 can be induced to contain high levels of KDOaldolase. The aldolase activity from this source was assayed accordingto Aminoff's method (Biochem. J. 1961, 81, 384). Two liters of culturecontained 10.2 U based on the degradation of KDO. This KDO activity is 4times and 8 times higher than the corresponding KDO activity fromEscherichia coli K-12 and Aerobacter colacae, respectively, as reportedby Gharambor (supra).

Partially purified KDO aldolase simply obtained by ammonium sulfateprecipitation (8.0 U/mL; 0.19 U/mg for degradation of KDO) was used insubstrate-specificity studies reported herein. The KDO aldolase employedfor the kinetic analysis reported herein, was further purified via DEAEsepharose, DEAE substituted beaded agarose, and phenyl sepharose, phenylsubstituted beaded agarose, column chromatography to a specific activityof 5.7 U/mg. The K_(m) for D-arabinose and V_(max) are 1.2M and 0.73U/mg, respectively. The unusually high concentration of K_(m) in thecondensation compared with that in the course of degradation (6×10⁻³ Mfor KDO) indicates that the enzyme may accept the open form of aldosesas acceptors in the aldol condensation. The enzymatic reaction favorsthe cleavage of KDO, with the equilibrium constant K_(eq)=[pyruvate][arabinose]/KDO=9×10⁻² M.

                  TABLE I                                                         ______________________________________                                        Relative Rates of Several Substrates for KDO Aldolase                         from Aureobacterium barkeri KDO-37-2                                                     relative               relative                                    substrate  rate.sup.a substrate   rate.sup.a                                  ______________________________________                                        D-arabinose                                                                              100        D-altrose   25                                                     N.D..sup.b,c                                                                             L-mannose   15                                          D-threose  128        L-arabinose N.D.                                        D-erythrose                                                                               93        D-xylose    N.D.                                        D-ribose    72        D-allose    N.D.                                        2-deoxy-D-ribose                                                                          71        D-glucose   N.D.                                        L-glyceraldehyde                                                                          36        D-mannose   N.D.                                        D-glyceraldehyde                                                                          23        L-fucose    N.D.                                        2-deoxy-2-fluoro-                                                                         46        N-acetyl    N.D.                                        D-arabinose           D-mannosamine                                           D-lyxose    35        N-acetyl    N.D.                                                              L-mannosamine                                           5-azido-2,5-                                                                              15        D-fructose  N.D.                                        dideoxy-D-ribose                                                              ______________________________________                                         .sup.a) Measured at pH 7.5 with 500 mM of sugar and 10 mM of pyruvate. Fo     detailed condition, see experimental. Specific activity based on              Darabinose is 0.2 U/mg; 1 U = 1 μmol KDO formed per min.                   .sup.b) Not detectable.                                                       .sup.c) Fluoropyruvate (10 mM) was used instead of pyruvate.             

Substrate Specificity

This enzyme exhibits a wide substrate specificity. Several 3-6 carbonsugars were accepted as substrates for the condensation. From theresults shown in Table I and FIG. 1, the structural requirements for thesugar for this enzyme are as follows. At C-2 position, although thealdolase prefers and S configuration, the difference is not significant[examples: between L- and D-glyceraldehyde; D-threose and D-erythrose;D-arabinose, D-ribose and 2-deoxy-D-ribose]. It is noteworthy that thisenzyme also accepts D-ribose as a good substrate (rel. V=72%), whilethat from E. coli or Aerobacter colacae poorly accepts this substrate(rel. V<5%), according to Gharambor (supra). Accordingly, the substratespecificity of KDO aldolase from Aureobacterium barkerei strain KDO-37-2with respect to D-ribose is considered favorable since it exceeds 7% ofthe corresponding substrate specificity of the enzyme with respect toD-arabinose. At C-3 position, this enzyme prefers an R-configuration[examples: comparison between D-arabinose and L-arabinose, D-lyxose andD-xylose]. Hexoses are generally not as good substrates as tetroses andpentoses, even in the case of D-altrose (rel. V=25%) and L-fucose (ratenot detectable), both being homoanalogs of the natural substrateD-arabinose. The reason that L-mannose is a better substrate thanD-mannose is because the former has the favorable 2R,3R configurationsand the latter has the unfavorable 2S,3S configurations. Finally,neither fluoropyruvate nor ketohexose was accepted by this enzyme.

The Aldol Condensation

The enzymatic synthesis of KDO on multi-mmol scales using 10 molarexcess of pyruvate worked well (e.g., 1 was obtained in 67% yield). Thesynthetic route is illustrated in FIG. 8A. The reagents employed in thissynthesis are as follows:

    ______________________________________                                        Step             Reagent or Enzyme                                            ______________________________________                                        (a)              KDO aldolase                                                 (b)              Ac.sub.2 O/py,DMAP                                           (c)              CH.sub.2 N.sub.2.                                            ______________________________________                                    

The yield of the enzymatic reaction is comparable to the highest oneobtained by the modified Cornforth synthesis (66%). The crystalline KDOammonium salt monohydrate was isolated in 37% yield: [α]²⁶ D +40.3° (c2.06, H₂ O) [lit. according to Unger: [α]²⁷ D +42.3° (c, 1.7, H₂ O),authentic sample from Sigma [α]²⁶ D +40.2° (c 2.06, H₂ O)]. (Unger: Adv.Carbohydr. Chem. Biochem. 1981, 38, 323.) The ¹ H NMR spectrum in D₂ Ois identical with that of an authentic sample, although it iscomplicated by the fact that KDO exists as an anomeric mixture ofpyranose and furanose forms, and readily cyclizes to the correspondinglactone in aqueous solution. The crystalline ammonium salt was furtherconverted to pentaacetate methyl ester derivative 2, whose ¹ H NMRspectrum was in good accordance with that reported previously andclearly shows the ⁵ C₂ -pyranose conformation (Table II).

                                      TABLE II                                    __________________________________________________________________________    .sup.1 H NMR Analysis of Pyranose, Furanose and 1 → 5 Lactone          Forms Observed                                                                in the Products Possessing a C-5 Axial Substituent in the Pyranose Form        ##STR1##                                                                                        ##STR2##                                                                                       ##STR3##                                                                                     ##STR4##                               chemical shifts (δ, ppm)                                                                    coupling constants (Hz)                       compound    H-3   H-3'          J.sub.3,3'                                                                        J.sub.3,4  J.sub.3',4                                                                        J.sub.3,5                  __________________________________________________________________________    1 (KDO, in D.sub.2 O)                                                         α-pyranose form                                                                     1.863 1.951         13.0                                                                              5.5        12.0                                                                              1.0                        β-pyranose form                                                                      2.373 1.735         11.7                                                                              5.0        11.7                                                                              1.0                        furanose form                                                                             2.275 2.351         13.5                                                                              7.5        7.5 --                         1' (in D.sub.2 O)                                                                         2.053 2.562         14.0                                                                              3.0        7.5 --                         2 (in CDCl.sub.3)                                                                         2.245 2.201         13.0                                                                              6.0        12.0                                                                              --                         11 (in D.sub.2 O)                                                             α-pyranose form                                                                     1.90-1.98           --  --         --  --                         furanose form                                                                             2.301 2.384         13.4                                                                              7.0        7.0 --                         11' (in D.sub.2 O)                                                                        2.072 2.576         14.2                                                                              3.1        7.3 --                         12 (in CDCl.sub.3)                                                                        2.339 2.972         14.9                                                                              2.4        9.5 0.6                        12' (in CDCl.sub.3)                                                                       2.10  2.80          15.0                                                                              2.5        9.0 --                         13 (in D.sub.2 O)                                                             α-pyranose form                                                                     1.873 1.984         13.0                                                                              5.2        11.9                                                                              --                         furanose form                                                                             2.284 2.341         13.1                                                                              6.4        6.4 --                         13' (in D.sub.2 O)                                                                        2.051 2.521         14.1                                                                              3.2        7.5 --                         14 (in CDCl.sub.3)                                                                        2.292 2.288         --  7.0        10.1                                                                              0.4                        __________________________________________________________________________

Several substrates with good or fair relative rate are shown to beemployable in the aldol condensation. The reactions with D-ribose and2-deoxy-D-ribose are illustrated in FIGS. 8B and 8C respectively. Thesereactions took place smoothly to give 3 (57% after derivation to 4) and5 (47% as 6), respectively. ¹ H NMR spectra of 3, 4, 5, and 6 clearlyshow a ⁵ C₂ pyranose form in both products (Table III). The ¹ H spectrumof 6 is shown in FIG. 2. It is noteworthy that in these cases, eventhough the relative rates are lower (72% for D-ribose and 71% for2-deoxy-D-ribose) than that of D-arabinose, TLC analysis of the reactionproducts showed no starting material left, whereas a substantial amountof starting material always remains in the reaction with D-arabinose. Itis suggested that formation of the pyranose form of 3 and 5, where allsubstituents are located in the stable orientation, further shifted theequilibrium toward condensation.

The products 7 (3-deoxy-D-arabino-2-heptulosonic acid, DAH, 39% as 8)and 9 (11% as 10) were also obtained from D-erythrose andD-glyceraldehyde, as illustrated in FIGS. 8D and 8E respectively. Theseyields indicate that this aldolase-catalyzed condensation is also usefulfor the synthesis of lower homologs of KDO. The phosphate of 7 (DAHP)plays an important role in the shikimate synthesis pathway in plants andmicroorganisms. The selected chemical shifts and coupling constants forthe ¹ H NMR spectra of products 3-10 are summarized in Table III.

                  TABLE III                                                       ______________________________________                                        .sup.1 H NMR Analysis of the α-Pyranose Form                            Observed in the Products Possessing a C-5 Equatorial Substituent               ##STR5##                                                                     chemical shift                                                                (δ, ppm)    coupling constants (Hz)                                     compound                                                                              H-3eq    H-3ax    J.sub.3eq,3ax                                                                       J.sub.3eq,4                                                                        J.sub.3ax,4                                                                         J.sub.3eq,5eq                      ______________________________________                                        3.sup.a 2.148    1.773    13.0  5.1  11.4  --                                 4.sup.b 2.559    2.010    13.5  5.2  11.6  --                                 5.sup.a 2.094    1.591    12.7  4.6  12.1  1.8                                6.sup.b 2.454    1.783    13.1  4.8  11.6  1.8                                7.sup.a 2.180    1.773    13.0  5.1  11.8  --                                 8.sup.b 2.658    2.087    13.6  5.2  11.4  --                                 9.sup.a 2.176    1.795    13.1  5.1  11.6  --                                 10.sup.b                                                                              2.618    1.948    13.5  5.2  11.2  --                                 ______________________________________                                         .sup.a) Measured in D.sub.2 O.                                                .sup.b) Measured in CDCl.sub.3.                                          

FIG. 8F illustrates the aldolase catalyzed condensation reaction can beemployed to produce product 11 from D-threose. Product 11 has a ¹ H NMRspectrum similar to that of KDO. The reaction with L-glyceraldehyde,illustrated in FIG. 8G afforded 13 (2-Keto-3- deoxy-L-gluconic acid,KDG), an enantiomer of D-KDG, whose phosphate (KDGP) is an intermediatein the Entner-Doudoroff pathway. (Entner, N.; Doudoroff, M. J. Biol.Chem. 1952, 196, 853.) The ¹ H NMR spectrum of 13 was very complicated(see experimental). To clarify the stereochemistry, preparation ofderivatives was attempted; however, the products were still difficult toidentify. The only isolable component from 11 was a bicyclic lactone.The structure was determined as 12 (FIG. 8F) by comparing its ¹ H NMRspectrum with that of the higher homolog 12', which had been obtainedfrom KDO and unambiguously characterized previously. (Charon, D.;Auzanneau, F.-I.; M erienne, C.; Szab o, L. Tetrahedron Lett. 1987, 23,1393.)

In its ¹ H NMR spectrum (Table II), a long range coupling between H-3and H-5 (0.6 Hz) indicates that the pyranose form of the product existsas a twisted boat conformation, and all of the coupling constants areconsistent with those observed in the case of 12'. It is interestingthat in the spectra of 11, 13 and KDO, a substantial proportion of thesimilar signals were observed, where one of the H-3 signal appears atvery low field (Table II). From these results, it is assumed that thebicyclic 1<<5 lactones 1', 11' and 13' form at nearly neutral pH. Theformation of 1<<7 lactone is excluded, since those signals were observedin the case of a hexulosonate 13' without any C-7 hydroxy group. Thehomologs prepared here also proceed through a spontaneous 1<<5 lactoneformation, as already proposed previously for KDO. (Menton, L. D., etal. Carbohydr. Res. 1980, 80, 295.) Compound 13 mainly exists as ⁵ C₂pyranose form as indicated in 14.

The reaction with L-mannose illustrated in FIG. 8H gave 15(3-deoxy-L-glycero-L-galacto-2-nonulosonic acid, L-KDN, 61% as 16),which is an enantiomer of D-KDN, a component in polysialoglycoproteinand ganglioside of rainbow trout eggs. (Lin, C.-H., et al., J. Am. Chem.Soc., in press; Nadano, D. et al. J. Biol. Chem. 1986, 261, 11550; andSong, Y., et al. J. Biol. Chem. 1991, 266, 21929). The optical rotation[[α]²⁵ D +26.3° (CHCl₃)] and ¹ H NMR spectrum of 16 were in goodaccordance with those of 16' [[α]²⁵ D -26.0° (CHCl₃)], which wasobtained via reaction with D-mannose catalyzed by sialic acid aldolase,as illustrated in FIG. 8I, except for the sign of rotation (Tetrahedron1990, 46, 201). The availability of both enantiomers of KDN may developnew analogs of sialyl oligosaccharides. (Ichikawa, Y., et al. Anal.Biochem. 1992, 202, 215.)

Finally, the aldol reaction with an unnatural sugar containing afluorine atom was conducted to give 18 (19% of 19). By comparing the ¹ HNMR spectra, the proportion of the β-isomer (10.71) of 18 was ca. 1.5times higher than that of KDO (6.9%), probably due to the absence offuranose and 1<<5 lactone forms. This result suggests that 18 might be agood substrate for CMP-KDO synthetase, since the enzyme accepts theunstable β-form of KDO as a substrate. (Kohlbrenner, W. E. and Fesik, S.W., J. Biol. Chem. 1985, 260, 14695.) We therefore synthesized 18 in alarger scale by combining the use of KDO aldolase and pyruvatedecarboxylase, which made the workup procedure much easier. Preliminarystudy using 18 toward CMP-KDO synthetase which had recently been clonedand over-expressed in this group showed that 18 was accepted to theenzyme.

Based on these results, the stereochemical course of the aldolcondensation catalyzed by this KDO aldolase is probably as follows: Theattack of pyruvate always takes place on the re face of the carbonylgroup of the substrates, a facial selection complementary to sialic acidaldolase reactions (si face attack). The stereochemical requirements ofsubstrates and the stereochemical course of the aldol condensation areindicated in FIG. 3. It is concluded that in general the enzyme acceptssubstrates with an R-configuration at C-3. The substrates with an Sconfiguration at C-2 is kinetically favored, while those with Rconfiguration at C-2 are thermodynamically favored to give a betteryield.

Synthesis of Decarboxylated Analogs

Decarboxylation of KDO and its analogs will yield the correspondingaldose derivatives. A synthetic route employing decarboxylation of KDOaldolase condensation products is illustrated in FIGS. 9A and 9B. Thereagents employed in these synthetic routes is as follows:

    ______________________________________                                        Step     Reagent                                                              ______________________________________                                        (a)      Ac.sub.2, DMAP/pyridine                                              (b)      CsCO.sub.3, BnBr/DMF                                                 (c)      H.sub.2,Pd--C/EtOH                                                   (d)      (COCl).sub.2 /toluene                                                (e)      17, DMAP/pyridine-toluene                                            (f)      t-BuSH, hv                                                           (g)      Me.sub.3 N═C═NEt (WSCI)-Cl, 17 (5 eq.) ,                              T--CuSH, DMAP, Et.sub.3 N, MS4A/Ch.sub.2 Cl.sub.2,                   ______________________________________                                                 hv                                                               

The aldodeoxyheptose structure is particularly interesting since anumber of heptoses are widely distributed in nature, some of which playimportant roles in metabolic pathways. Barton's radical-mediateddecarboxylation of the penta-O-acetyl derivative 20a obtained from thecorresponding benzyl ester 20b seems to be the most straightforwardroute to the desired heptose derivative 21. (e.g., Crich, D. and Lim, L.B. L. J. Chem. Soc. Perkin I 1991, 2209 and Auzanneau, F.-I. et al.Carbohydr. Res. 1990, 201, 337.)

There have recently been growing interests in the synthesis ofphysiologically active carbohydrate- and nucleic acid-related compoundsvia anomeric radical intermediates. It appears to us thatradical-mediated reaction stabilized both electron withdrawing anddonating group (capto-dative effect), e.g. Viehe, H. G. et al. Acc.Chem. Res. 1985, 18, 148.) at anomeric position [--C()(OAc)O-type] israre (only a few related examples [eg. --C()(CO₂ Me)O-- type,--C()(CHF₂)O-- type] are known), while examples in the case of simpleanomeric radical [--C()(H or R)O-- type] and the one bearing twoelectron donating oxygen atom [--C()(OR))-- type] have been extensivelystudies. (e.g. Crich, D. and Lim, L. B. L. J. Chem. Soc. Perkin I 1991,2205 and J. Chem. Soc. Perkin I 1991, 2209; Schmidt, R. et al.Tetrahedron Lett. 1988, 29, 3643; Myrvold, S. et al. J. Am. Chem. Soc.1989, 111, 1861; Motherwell, W. B. et al. Synlett. 1989, 68; and Samadi,M. J. Med. Chem. 1992, 35, 63.) The radical intermediate was formed bythe thermal decomposition of the thiohydroxamate 20c generated in situfrom the corresponding acid chloride and 22 in the presence ofazobisisobutyronitrile (AIBN). The subsequent trapping with tributyltinhydride resulted in only a disappointing (less than 2%) yield of 21. Theyield was, however, dramatically improved to 68% by irradiation withwhite light in the presence of t-butylmercaptane.

The ¹ H NMR spectrum of 21 clearly shows the exclusive β-anomer (δ 5.75,dd, J₁,2eq =3.0, J₁,2ax =10.0 Hz, H-1), indicating that the abstractionof hydrogen atom from t-butylmercaptane took place at the bottom side ofthe six-membered ring. The proposed mechanism for the exclusiveformation of β-isomer is as follows. The stable conformer of the radicalintermediate which is stabilized both by the electron-donating andwithdrawing effects is supposed to be in a plane form as depicted inFIGS. 9A and 9B, which allows the maximum interaction between theone-electron p orbital and the lone pair electrons on the adjacent ringoxygen. t-Butylmercaptane is easily accessible from the bottom side,while the approach from the top side is sterically hindered by thehydrogen and acetoxy groups. This explanation in terms of kineticcontrol is well matched with the thermodynamic stability of theβ-product.

The radical process was also applied to the synthesis of thedecarboxylated analog of N-acetylneuraminic acid. It turned out,however, that all attempts for the synthesis of the acyl chlorideresulted in a complex mixture, even from fully protected peracetate form23a of sialic acid, because NHAc proton still has a substantialreactivity to chlorinating reagents. The direct formation ofthiohydroxamate 23b was also found to be difficult because of theinherent steric hindrance around carbonyl group in the startingmaterial. Through an extensive examination of the reaction conditions,it was found that the combination of ethyl(diethylamino)propylcarbodiimide hydrochloride (WSCI-Cl, 1.5 eq) andexcess of 22 (5.0 eq) worked well for the in situ formation anddegradation of thiohydroxamate, to give 24 (27% yield from 23a). Thiscondition has the advantage that the reaction can be carried out in onestep. The newly formed product was exclusively an α-anomer where the OAcgroup is located in the equatorial orientation, consistent with theresult obtained in the decarboxylation of KDO derivative. (Haverkamp, J.et al. Eur. J. Biochem. 1982, 122, 305.)

PREPARATION OF EXAMPLES

General

Optical rotations were measured on Perkin-Elmer 241 spectrophotometer UVand visible spectra were recorded on a Beckmann DU-70 spectrometer. ¹ Hand ¹³ C NMR spectra were recorded at 400 and 500 MHz on Bruker AMX-400and AMX-500 spectrometer. High-resolution mass spectra (HRMS) wererecorded on a VG ZAB-ZSE mass spectrometer under fast atom bombardment(FAB) conditions. Column chromatography was carried out with silica gelof 70-230 mesh. Preparative TLC was carried out on Merck Art. 5744 (0.5mm).

Isolation of the Microorganism

Aureobacterium barkerei containing high levels of KDO aldolases wasselected with the S medium containing 0.25% of synthetic KDO mixture ascarbon source (20 mL) in serum bottles (158 mL) and incubated at 37° C.for 2 days with shaking (250 r.p.m.). (McNicholas, P. A. et al.Carbohydr. Res. 1986, 146, 219 and Shirai, R.; and Ogura, H. TetrahedronLett. 1989, 30, 2263.) The bottles which showed turbidity weretransferred to the same fresh medium. After several transfers, thecultures were plated on the S medium agar plates (1.5% agar) containing0.25% of synthetic KDO mixture. The isolated colonies were transferredto the liquid medium as described above. To confirm the utilization ofKDO, the disappearance in the medium was monitored by TLC as describedin the synthesis of KDO. The cultures which showed the utilization ofKDO were harvested by centrifugation and resuspended in 50 mM phosphatebuffer (pH 7.0). The cell suspension was incubated with 1% (w/v) ofauthentic KDO (from Sigma) at 37° C. overnight to confirm thedegradation of KDO by TLC. The cultures were then replated on LB agarplates to ensure the purity of the culture.

Preparation of the Enzyme

With one slight modification, the incubation was carried out accordingto the procedure reported by Gharambor (supra). The ingredients of themedium were as follows: NH₄ Cl (5 g), K₂ SO₄ (1 g), MgSO₄ •7H₂ O (200mg), CaCl₂ (20 mg), FeSO₄ •7H₂ O (1 mg), yeast extract (1 g), Na₂ HPO₄•7H₂ O (10 g), and KH₂ PO₄ (3 g) in distilled water (1 L), at pH 7.2. Toa 50 mL of this medium in a 100 mL Erlenmeyer flask, were addedD-glucose (40% solution in water, 25 μL) and KDO (100 mg, 0.2%), and aloopful of Aureobacterium barkerei KDO-37-2 was incolutated. The flaskwas shaken at 250 r.p.m. on a gyrorotary shaker at 30° C. for 16 h. Theseed culture thus obtained was poured into the 1950 mL of the sameincubation medium containing KDO (3.9 g). The mixture was divided andpoured into two of 2.8 L Erlenmeyer flasks. The flasks were shaken at250 r.p.m. at 30° C. for 24 h. The growth of microorganism was estimatedby OD at 600 nm to be 1.90. The cells were harvested at 10,000×g for 30min at 4° C. and washed with 50 mM potassium-sodium phosphate buffer (pH7.5). The collected cells were then resuspended in the same buffersolution (20 mL) and disrupted by French-pressure apparatus (at 16,000lb/in). The cell debris were removed by centrifuge at 23,000×g for 1 hat 4° C. to give the supernatant (ca. 20 mL) as the crude enzymepreparation. The enzyme activity was determined to be 1.45 U/mL for thedegradation of KDO according to the method of Aminoff (Biochem. J. 1961,81, 384). Ammonium sulfate precipitation between 45-75% saturation wascollected and dialyzed in phosphate buffer (2 L; 100 mM, 1 mM ofdithiothreitol, 2 L) to give partially purified enzyme (13.5 mL, 1.73U/mL for KDO degradation), according to the method of Kim (J. Am. Chem.Soc. 1988, 110, 6481).

Kinetic Measurements

The rates for aldolase-catalyzed reactions were obtained by measuringthe amount of remaining pyruvate, according the method of Kim (supra).The reactions were carried out in 0.1M phosphate buffer (pH 7.5)containing: varied concentrations of pyruvate, 2.0, 3.33, 5, and 10 mM;varied concentrations of D-arabinose, 0.2, 0.25, 0.33, and 0.50M in 0.5mL of solution. Each solution was incubated at 37° C. Periodically, asmall aliquot (25-100 μL) was withdrawn and mixed with an assay solution(1.4 mL) containing 0.1M phosphate (pH 7.5) buffer, 0.3 mM NADH, and20-30 U of L-lactate dehydrogenase. The decrease in absorbance at 340 nmwas measured and converted into the amount of the unreacted pyruvateusing 6220M⁻¹ cm⁻¹ for the molecular absorbance of NADH. The kineticparameters were obtained from the Lineweaver-Burk plots.

For the relative rate measurements, the concentration of pyruvate(fluoropyruvate) and sugar were fixed at 10 mM and 0.5M, respectively.Other conditions were the same as above.

Example 1 Ammonium 3-deoxy-α-D-manno-2-octulosonate monohydrate (KDOammonium salt monohydrate, 1)

D-Arabinose (250 mg, 1.67 mmol), sodium pyruvate (1.83 g, 16.7 mmol),dithiothreitol (1.5 mg), NaN₃ (2% solution in water, 100 μL), NaHPO₄•7H₂ O (53 mg), and KH₂ PO₄ (13 mg) were added to the KDO aldolase (5.1U, 10 mL). The pH was adjusted to 7.5 and the mixture was stirred underN₂ at 30° C. for 3 days. The product was purified by treatment with aDowex-1 resin column (bicarbonate form) eluted with a linear gradientfrom 0 to 0.25M of ammonium bicarbonate. KDO ammonium salt was furtherpurified by Biogel P-2 column. The fraction eluted with H₂ O containingKDO was collected and its total amount was estimated to be 1.11 mmol(67%) by Aminoff's assay (supra). The residue after lyophilization wasrecrystallized from aqueous ethanol to give colorless plates (168 mg,37% from D-arabinose): mp 123°-125° C. (decomposition) [lit. accordingto Hershberger: mp 121°-123° C., authentic sample from sigma mp123°-125° C. (decomposition)]; [α]²⁶ D +40.3° (c 2.06, water) [lit.according to Hershberger: [α]²⁷ D +42.3° (c 1.7, water), authenticsample from Sigma [α]²⁶ D +40.2° (c 2.03, water)]. Its ¹ H NMR spectrumin D₂ O was identical with that of an authentic sample. (Hershberger: J.Biol. Chem. 1968, 243, 1585.) A small portion was converted topentaacetate methyl ester derivative 2: ¹ H NMR (CDCl₃) δ 1.994 (3H, s,acetyl), 1.998 (3H, s, acetyl), 2.045 (3H, s acetyl), 2.108 (3H, s,acetyl), 2.139 (3H, s, acetyl), 2.201 (1H, dd, J_(3ax),4 =12.0,J_(3ax),3eq =13.0 Hz, H-3ax), 2.245 (1H, dd, J_(3eq),4 =6.0, J_(3eq),3ax=13.0 Hz, H-3eq), 3.810 (3H, s, COOCH₃), 4.113 (1H, dd, J_(8'),7 =12.5,J_(8'),8 =12.5 Hz, H-8'), 4.173 (1H, dd, J₆,5 =1.3, J₆,7 =9.5 Hz, H-6),4.475 (1H, dd, J₈,7 =4.0, J₈,8' =12.5 Hz, H-8), 5.220 (1H, ddd, J₇,8=4.0, J₇,6 =9.5, J₇,8' =12.5 Hz, H-7), 5.322 (1H, ddd, J₄,5 =3.0, J₄,3eq=6.0, J₄,3eq =6.0, J₄,3ax =12.0 Hz, H-4), 5.385 (1H, dd, J₅,6 =1.3, J₅,4=3.0 Hz, H-5). The ¹ H NMR spectrum was in good accordance with thatreported previously by Unger (Adv. Carbohydr. Chem. Biochem. 1981, 38,323).

Example 2 Methyl2,4,5,7,8-penta-O-acetyl-3-deoxy-α-D-altro-2-octulosonate (4)

In the same manner as described for the preparation of 1, the product 3(as ammonium salt) was prepared from D-ribose (0.33 mmol): ¹ H NMR (D₂O) δ 1.773 (1H, dd, J_(3ax),4 =11.9, J_(3ax),3eq =13.0 Hz, H-3ax), 2.148(1H, dd, J_(3eq),4 =5.1, J_(3eq),3ax =13.0 Hz, H-3eq), 3.500 (1H, dd,J₅,4 =9.1, J₅,6 =10.0 Hz, H-5), 3.745 (1H, dd, J₈,7 =7.3, J₈,8' =12.1Hz, H-8), 3.789 (1H, dd, J_(8'),7 =3.7, J_(8'),8 =12.1 Hz, H-8'), 3.809(1H, dd, J₆,7 =2.8, J₆,5 =10.0 Hz, H-6), 3.901 (1H, ddd, J₄,3eq =5.1,J₄,5 =9.1, J₄,3ax =11.9 Ha, H-4), 4.004 (1H, dd, J₇,6 =2.8, J₇,8' =3.7,J₇,8 =7.3 Hz, H-7). This was converted to 4 by the successive treatmentwith acetic anhydride-pyridine-DMAP (see also the preparation of 20b)and etherial diazomethane solution. The product was purified with silicagel preparative TLC to afford 4 (87.7 mg, 57% from D-ribose) as an oil,[α]²⁵ D +70.9° (c 0.81, CHCl₃); ¹ H NMR (CDCl₃) δ 2.010 (1H, dd,J_(3ax),4 =11.6, J_(3ax),3eq =13.5 Hz, H-3ax), 2.030 (3H, s, acetyl),2.050 (3H, s, acetyl), 2.064 (3H, s, acetyl), 2.105 (3H, s, acetyl),2.154 (3H, s, acetyl), 2.559 (1H, dd, J_(3eq),4 =5.2, J_(3eq),3ax =13.5Hz, H-3eq), 3.793 (3H, s, COOCH₃), 4.084 (1H, dd, J₆,7 =3.2, J₆,5 =10.3Hz, H-6), 4.241 (1H, dd, J₈,7 =7.0, J₈,8' =12.0 Hz, H-8), 4.415 (1H, dd,J_(8'),7 =4.0 J_(8'),8 =12.0 Hz, H-8'), 5.110 (1H, dd, J₅,4 =9.3, J₅,6=10.3 Hz, H-5), 5.169 (1H, ddd, J₇,6 =3.2, J₇,8' =4.0, J₇,8 =7.0 Hz,H-7), 5.271 (1H, ddd, J₄,3eq =5.2, J₄,5 =9.3, J₄,3ax =11.6 Hz, H-4); ¹³C NMR (CDCl₃) δ 20.52, 20.56, 20.56, 20.67, 20.67, 35.47, 53.12, 61.23,68.33, 68.96, 69.85, 71.98, 96.66, 166.21, 167.94, 169.52, 169.85,169.89, 170.38. HRMS (M+Cs⁺) calcd C₁₉ H₂₆ O₁₃ Cs 595.0428, found595.0428.

Example 3 Methyl2,4,7,8-tetra-O-acetyl-3,5-dideoxy-α-D-manno-2-octulosonate (6)

In the same manner as 3, the product 5 (as ammonium salt) was preparedfrom 2-deoxy-D-ribose (0.33 mmol): ¹ H NMR (D₂ O) δ 1.400 (1H, ddd,J_(5ax),4 =11.9, J_(5ax),6 =11.9, J_(5ax),5eq =12.3 Hz, H-5ax), 1.591(1H, dd, J_(3ax),4 =12.1, J_(3ax),3eq =12.7 Hz, H-3ax), 2.009 (1H, dddd,J_(5eq),3eq =1.8, J_(5eq),6 =2.2, J_(5eq),4 =4.6, J_(5eq),5ax =12.3 Hz,H-5eq), 2.094 (1H, ddd, J_(3eq),5eq =1.8, J_(3eq),4 =4.6, J_(3eq),3ax=12.7 Hz, H-3eq), 3.398 (1H, dd, J₈,7 =7.1, J₈,8' =11.8 Hz, H-8), 3.588(1H, dd, J_(8'),7 =4.1, J_(8'),8 =11.8 Hz, H-8'), 3.786 (1H, ddd, J₇,8'=4.1, J₇,6 =4.6, J₇,8 =7.1 H8, H-7), 3.945 (1H, ddd, J₆,5eq =2.2, J₆,7=4.6, J₆,5ax =11.9 Hz, H-6), 4.112 (1H, dddd, J₄,3eq =4.6, J₄,5eq =4.6,J₄,5ax =11.9, J₄,3ax =12.1 Hz, H-4). This was converted to 6 (62.2 mg,,47% from 2-deoxy-D-ribose): [α]²⁵ D +86.0° (c 0.56, CHCl₃); ¹ H NMR(CDCl₃) δ 1.488 (1H, ddd, J_(5ax),4 =12.0, J_(5ax),6 =12.0, J_(5ax),5eq=12.7 Hz, H-5ax), 1.783 (1H, dd, J_(3ax),4 =11.6, J_(3ax),3eq =13.1 Hz,H-3ax), 2.045 (3H, s, acetyl), 2.054 (3H, s, acetyl), 2.070 (3H, s,acetyl), 2.123 (3H, s, acetyl), 2.177 (1H, dddd, J_(5eq),3eq =1.8,J_(5eq),6 =2.2, J_(5eq),4 =4.7, J_(5eq),5ax =12.7 Hz, H-5eq), 2.454 (1H,ddd, J_(3eq),5eq =1.8, J_(3eq),4 =4.8, J_(3eq),3ax =13.1 Hz, H-3eq),3.782 (3H, s, COOCH₃), 4.034 (1H, ddd, J₆,5eq =2.2, J₆,7 =7.6, J₆,5ax=12.0 Hz, H-6), 4.169 (1H, dd, J₈,7 =5.1, J₈,8' =12.2 Hz, H-8), 4.457(1H, dd, J_(8'),7 =2.8, J_(8'),8 =12.2 Hz, H-8'), 5.093 (1H, ddd, J₇,8'=2.8, J₇,8 =5.1, J₇,6 =7.6 Hz, H-7), 5.186 (1H, dddd, J₄,5eq =4.7,J₄,3eq =4.8, J₄,3ax =11.6, J₄,5ax =12.0 Hz, H-4); ¹³ C NMR (CDCl₃) δ20.56, 20.56, 20.73, 20.96, 32.21, 36.03, 52.96, 61.82, 65.72, 69.00,71.96, 97.61, 167.02, 167.96, 169.81, 170.06, 170.32. HRMS (M+Cs⁺) calcdC₁₇ H₂₄ O₁₁ Cs 537.0373, found 537.0373.

Example 4 Methyl2,4,5,7-tetra-O-acetyl-3-deoxy-α-D-arabino-2-heptulosonate (8)

7: ¹ H NMR (D₂ O) δ 1.773 (1H, dd, J_(3ax),4 =11.8, J_(3ax),3eq =13.0Hz, H-3ax), 2.180 (1H, dd, J_(3eq),4 =5.1, J_(3eq),3ax =13.0 Hz, H-3eq),3.433 (1H, dd, J₅,4 =9.2, J₅,6 =9.5 Hz, H-5), 3.744 (1H, ddd, J₆,7 =3.5,J₆,7' =3.5, J₆,5 =9.5 Hz, H-6) 3.807 (1H, m, H-7), 3.812 (1H, m, H-7'),3.930 (1H, ddd, J₄,3eq =5.1, J₄,5 =9.2, J₄,3a =11.8 Hz, H-4).

8: (50.0 mg, 39% from 0.33 mmol of D-erythrose): [α]²⁵ D +54.0° (c 0.50,CHCl₃); ¹ H NMR (CDCl₃) δ 2.034 (3H, s, acetyl), 2.053 (3H, s, acetyl),2.087 (3H, s, acetyl), 2.087 (1H, dd, J_(3ax),4 =11.4, J_(3ax),3eq =13.6Hz, H-3ax), 2.173 (3H, s, acetyl), 2.658 (1H, dd, J_(3eq),4 =5.2,J_(3eq),3ax =13.6 Hz, H-3eq), 3.808 (3H, s, COOCH₃), 4.058 (1H, dd, J₆,7=2.3, J₆,7' =4.3, J₆,5 =10.2 Hz, H-6), 4.100 (1H, J₇,6 =2.3, J₇,7' =12.4Hz, H-7), 4.355 (1H, J_(7'),6 =4.3, J_(7'),7 =12.4 Hz, H-7'); ¹³ C NMR(CDCl₃) δ 20.65, 20.76, 20.76, 20.84, 35.58, 53.31, 61.69, 68.16, 68.37,71.51, 97.29, 166.41, 168.43, 169.61, 170.13, 170.77. HRMS (M+Cs⁺) calcdC₁₆ H₂₂ O₁₁ Cs 523.0216, found 523.0216.

Example 5 Methyl 2,4,5-tri-O-acetyl-2-keto-3-deoxy-α-D-galactonate (10)

9: ¹ H NMR (D₂ O) δ 1.795 (1H, dd, J_(3ax),4 =11.6, J_(3ax),3eq =13.1Hz, H-3ax), 2.176 (1H, dd, J_(3eq),4 =5.1, J_(3eq),3ax =13.1 Hz, H-3eq),3.60-3.65 (2H, m), 3.77-3.91 (2H, m).

10: (11.0 mg, 11% from 0.33 mmol of D-glyceraldehyde): [α]²⁵ D +31.8° (c1.10, CHCl₃); ¹ H NMR (CDCl₃) δ 1.948 (1H, dd, J_(3ax),4 =11.2,J_(3ax),3eq =13.5 Hz, H-3ax), 2.055 (3H, s, acetyl), 2.059 (3H, s,acetyl), 2.170 (3H, s, acetyl), 2.618 (1H, dd, J_(3eq),4 =5.2,J_(3eq),3ax =13.5 Hz, H-3eq), 3.629 (1H, dd, J_(6ax),5 =10.6,J_(6ax),6eq =11.3 Hz, H-6ax), 3.809 (3H, s, COOCH₃), 4.149 (1H, dd,J_(6eq),5 =5.7, J_(6eq),6ax =11.3 Hz, H-6eq), 5.049 (1H, ddd, J₅,6eq=5.7, J₅,4 =9.5, J₅,6ax =10.6 Hz, H-5), 5.320 (1H, ddd, J₄,3eq =5.2,J₄,5 =9.5, J₄,3ax =11.2 Hz, H-4); ¹³ C NMR (CDCl₃) δ 20.67, 20.72,20.89, 35.81, 53.25, 62.17, 67.66, 68.49, 96.80, 166.96, 168.50, 169.84,170.05. HRMS (M+Cs⁺) calcd C₁₃ H₁₈ O₉ Cs 451.0005, found 451.0005.

Example 6 2,4,7-Tri-O-acetyl-3-deoxy-α-D-lyxo-2-heptulosonic acid 1<<5lactone (12)

11: ¹ H NMR (D₂ O) δ 1.90-1.98 (m, H-3 of the major component); a minorpair of H-3protons: 2.072 (dd, J₃,4 =3.1, J₃,3' =14.2 Hz, H-3), 2.576(dd, J_(3'),4 =7.3, J_(3'),3 =14.2 Hz, H-3'); another minor pair of H-3protons: 2.301 (dd, J=7.0, 13.4 Hz), 2.384 (dd, J=7.0, 13.4 Hz);3.60-3.95 (m), 3.95-4.20 (m), 4.48-4.52 (m).

12: (1.9 mg): ¹ H NMR (CDCl₃) δ 2.096 (3H, s, acetyl), 2.127 (3H, s,acetyl), 2.180 (3H, s, acetyl), 2.339 (1H, ddd, J₃,5 =0.6, J₃,4 =2.4,J₃,3' =14.9 Hz, H-3) 2.972 (1H, dd, J_(3'),4 =9.4, J_(3'),3 =14.9 Hz,H-3'), 4.180 (1H, ABX type, J₆,7 =5.6, J₆,7' =9.9 Hz, H-6), 4.28-4.35(2H, m, ABX type, H-7, H-7'), 4.904 (1H, d, J₅,4 =2.0 Hz, H-5), 5.164(1H, ddd, J₄,5 =2.0, J₄,3 =2.4, J₄,3' =9.4 Hz, H-4). HRMS (M+Cs⁺) calcdC₁₃ H₁₆ O₉ Cs 448.9849, found 448.9858.

Example 7 Methyl 2,4,5-tri-O-acetyl-2-keto-3-deoxy-α-L-gluconate (14)

13 (L-KDG): ¹ H NMR (D₂) A major pair of H-3-protons: δ 1.873 (dd,J_(3eq),4 =5.2, J_(3eq),3ax =13.0 Hz, H-3eq), 1.984 (dd, J_(3ax),4=11.9, J_(3ax),3eq =13.0 Hz, H-3ax); a minor pair of H-3 protons: 2.051(dd, J₃,4 =3.2, J₃,3' =14.1 Hz, H-3), 2.521 (dd, J_(3'),4 =7.5, J_(3'),3=14.1 Hz, H-3'); a minor H-3 proton (² C₅ β-pyranose form is suggested):2.167 (dd, J₃,4 =4.0, J₃,3' =13.7 Hz), in this case the H-3' protoncould not be specified by overlapping of the signals; another minor pairof H-3 protons: 2.284 (dd, J=6.4, 13.1 Hz), 2.341 (dd, J=6.4, 13.1 Hz);3.60-4.10 (m), 4.15-4.20 (m), 4.30-4.40 (m).

14: (2.0 mg): ¹ H NMR (CDCl₃) δ 2.034 (3H, s, acetyl), 2.150 (3H, s,acetyl), 2.152 (3H, s, acetyl), 2.288 (1H, d, J_(3ax),4 =10.1 Hz,H-3ax), 2.292 (1H, dd, J_(3eq),5 =0.4 Hz, J_(3eq),4 =7.0 Hz, H-3eq),3.830 (3H, s, COOCH₃), 3.999 (1H, dd, J_(6eq),5 =1.5, J_(6eq),6ax =13.2Hz, H-6eq), 4.092 (1H, dd, J_(6ax),5 2.0, J_(6ax),6eq =13.2 Hz, H-6ax),5.251 (1H, dddd, J₅,3eq =0.4, J₅,6eq =1.5, J₅,6ax =2.0, J₅,4 =2.7, Hz,H-5), 5.313 (1H, ddd, J₄,5 =2.7, J₄,3eq =7.0, J₄,3ax =10.1 Hz, H-4).HRMS (M+Na⁺) calcd C₁₃ H₁₈ O₉ Na 341.0849, found 341.0849.

Example 8 Methyl2,4,5,7,8,9-hexa-O-acetyl-3-deoxy-β-L-glycero-L-galacto-nonulosonate(16)

15 (L-KDN): ¹ H NMR (D₂ O) δ 1.773 (1H, dd, J_(3ax),4 =11.8, J_(3as),3eq=12.9 Hz, H-3ax), 2.168 (1H, dd, J_(3eq),4 =5.1, J_(3eq),3ax =11.8 Hz,H-3eq), 3.579 (1H, dd, J₅,4 =9.3, J₅,6 =9.9 Hz, H-5), 3.654 (1H, dd,J₉,8 =6.3, J₉,9' =11.8 Hz, H-9), 3.766 (1H, ddd, J₈,9' =2.6, J₈,9 =6.3,J₈,7 =9.0 Hz, H-8), 3.831 (1H, dd, J₇,6 =1.1, J₇,8 =9.0 Hz, H-7), 3.873(1H, dd, J_(9'),8 =2.6, J_(9'),9 =11.8 Hz, H-9'), 3.925 (1H, dd, J₆,7=1.1, J₆,5 =9.9 Hz, H-6), 3.971 (1H, ddd, J₄,3eq =5.1, J₄,5 =9.3, J₄,3ax=11.8 Hz, H-4).

16 (108.3 mg, 61% from 0.33 mmol of L-mannose): [α]²⁵ D +26.3° (c 1.14,CHCl₃); ¹ H NMR (CDCl₃) δ 2.084 (1H, dd, J_(3ax),4 =11.6, J_(3ax),3eq=13.6 Hz, H-3ax), 2.013 (3H, s, acetyl), 2.024 (3H, s, acetyl), 2.040(3H, s, acetyl), 2.069 (3H, s, acetyl), 2.115 (3H, s, acetyl), 2.157(3H, s, acetyl), 2.625 (1H, dd, J_(3eq),4 =5.3, J_(3eq),3ax =13.6 Hz,H-3eq), 3.790 (3H, s, COOCH₃), 4.141 (1H, dd, J₉,8 =5.8, J₉,9' =12.6 Hz,H-9), 4.186 (1H, dd, J₆,7 =2.3, J₆,5 =10.3 Hz, H-6), 4.440 (1H, dd,J_(9'),8 =2.5, J_(9'),9 =12.6 Hz, H-9'), 4.975 (1H, dd, J₅,4 =9.6, J₅,6=10.3 Hz, H-5), 5.150 (1H, ddd, J₈,9' =2.5, J₈,9 =5.8, H₈,7 =6.3 Hz,H-8), 5.264 (1H, ddd, J₄,3eq =5.3, J₄,5 =9.6, J₄,3ax =11.6 Hz, H-4),5.396 (1H, dd, J₇,6 =2.3, J₇,8 =6.3 Hz, H-7); ¹³ C NMR (CDCl₃) δ 20.46,20.48, 20.58, 20.58, 20.67, 35.32, 53.06, 61.67, 66.68, 67.21, 68.57,70.00, 71.27, 97.14, 165.91, 168.03, 169.46, 169.57, 169.80, 169.96,170.41. HRMS (M+Cs⁺) calcd. C₂₂ H₃₀ O₁₅ Cs 667.0639, found 667.0639.

16': [α]²⁵ D -26.0° (c 1.00, CHCl₃). The ¹ H NMR spectrum was identicalwith that of 16.

Example 9 2-Deoxy-2-fluoro-D-arabinose (17b)

To a solution of a tribenzoate 17a (available from Pfanstiehl Co., 500mg, 1.08 mmol) in ethanol (5 mL) was added 10N NaOH aqueous solution(485 μL, 1.5 eq of each OBz group, total 4.5 eq) at room temperature.After 15 min, H₂ O (10 mL) and ethanol (5 mL) were added and the mixturewas stirred and heated to 50° C. to dissolve the precipitated sodiumbenzoate. The mixture was further stirred for 1 h at room temperature.After ethanol was evaporated in vacuo, the residue was dissolved in H₂ Oand Dowex 50W-X8 (H⁺ form) was added to acidify the mixture. Theprecipitated benzoic acid was filtered off, and the filtrate was treatedwith Dowex 1-X8 (HCO₃ ⁻ form) and filtered, then concentrate in vacuo togive 17b as colorless syrup (153 mg, 94%); ¹ H NMR (D₂ O) δ 3.60-4.20(4H, m), 4.337 (ddd, J₂,1 =7.7, J₂,3 =9.3, J₂,F =51.8 Hz, H-2 ofβ-anomer), 4.666 (ddd, J₂,1 =3.7, J₂,3 =9.5, J₂,F =49.5 Hz, H-2 ofα-anomer), 4.763 (dd, J₁,F =3.3, J₁,2 =7.7, H-1 of β-anomer), 5.434 (dd,J₁,F =1.5, J₁,2 =3.7 Hz, H-1 of α-anomer). This anomeric mixture wasused in the next step without further purification.

Example 10 Methyl2,4,7,8-tetra-O-acetyl-3,5-dideoxy-5-fluoro-α-D-manno-2-octulosonate(19)

18: ¹ H NMR (D₂ O) δ 1.814 (dd, J_(3ax),3eq =12.4, J_(3ax),4 =12.4 Hz,H-3ax of β-anomer), 1.988 (1H, ddd, J_(3eq),5 =0.8, J_(3eq),4 =5.6,J_(3eq),3ax =12.9 Hz, H-3eq of α-anomer), 2.058 (1H, dd, J_(3ax),4=11.8, J_(3ax),3eq =12.9 Hz, H-3ax of α-anomer), 2.461 (ddd, J_(3q),5=0.8, J_(3eq),4 =5.3, J_(3eq),3ax =12.4 Hz, H-3eq of β-anomer), 3.663(1H, dd, J₈,7 =5.4, J₈,8' =12.1 Hz, H-8), 3.828 (1H, dd, J_(8'),7 =2.4,J_(8'8) =12.1 Hz, H-8'), 3.80-3.95 (2H, m), 4.182 (1H, dddd, J₄,5 =2.4,J₄,3eq =5.6, J₄,3ax =11.8, J₄,F =30.5 Hz, H-4), 4.957 (1H, ddd, J₅,3eq=0.8, J₅,4 =2.4, J₅,F =50.9 Hz, H-5).

19 (25.3 mg, 18% from 0.33 mmol of 17b): [α]²⁵ D +96.4° (c 2.53, CHCl₃);¹ H NMR (CDCl₃) δ 2.043 (3H, s, acetyl), 2.067 (3H, s, acetyl), 2.131(3H, s, acetyl), 2.137 (3H, s, acetyl), 2.271 (1H, dd, J_(3ax),4 =11.5,J_(3ax),3eq =13.3 Hz, H-3ax), 2.319 (1H, dd, J_(3eq),4 =5.9, J_(3eq),3ax=13.3 Hz, H-3eq), 3.805 (3H, s, COOCH₃), 4.073 (1H, dd, J₆,7 =9.5, J₆,F=27.8 Hz, H-6), 4.154 (1H, dd, J₈,7 =3.5, J₈,8' =12.5 Hz, H-8), 4.601(1H, dd, J_(8'),7 =2.2, J_(8'),8 =12.5 Hz, H-8'), 4.827 (1H, dd, J₅,4=2.1, J₅,F =50.9 Hz, H-5), 5.240 (1H, dddd, J₄,5 =2.1, J₄,3eq =5.9,J₄,3ax =11.5, J₄,F =21.3 Hz, H-4), 5.288 (1H, ddd, J₇,8' =2.2, J₇,8=3.5, J₇,6 =9.5 Hz, H-7); ¹³ C NMR (CDCl₃) δ 20.56, 20.56, 20.71, 20.83,30.60, 53.18, 61.46, 66.45, (d, J_(C),F =17.8 Hz), 67.89 (d, J_(C),F=4.1 Hz), 69.60 (d, J_(C),F =18.2 Hz, 83.02 (d, J_(C),F =186.2 Hz),97.04, 166.49, 167.75, 169.14, 170.18, 170.20. HRMS (M+Cs⁺) calcd C₁₇H₂₃ O₁₁ FCs 555.0279, found 555.0288.

Example 11 Larger scale synthesis of 18

Fluorosugar 17b (340 mg, 2.25 mmol), sodium pyruvate (2.074 g, 28.9mmol), dithiothreitol (1.7 mg), NaN₃ (2.3 mg), phosphate buffer (pH 7.5,50 mM, 1.12 mL) was added to the enzyme solution (3.0 mL, 24 U). Afterthe pH was adjusted to 7.5, the volume was made up to 10.0 mL. Themixture was stirred under N₂ at room temperature for 7 days. The pH waslowered to 2.5 by addition of Dowex 50W-X8 (H⁺ form) and the mixture waskept at 0° C. for 1 h. The precipitate was removed by centrifugation at23,000×g for 1 h at 4° C. Before the anion-exchange resin treatment, theexcess pyruvate was removed as follows. The mixture was diluted to 80 mLand the pH was adjusted to 6.5 by the addition of 2N aqueous ammoniasolution. The antifoam (Antifoam AF emulsion, Dow-Corning Nakaraitesque,10% emulsion in water, 0.32 mL) and pyruvate decarboxylase (Sigma P6810, 0.2 mL, 12.5 U) was added and the mixture was stirred at roomtemperature with bubbling of N₂ (1.5 L/min). The pH was monitored andoccasionally adjusted between 6.0 and 6.5, by addition of Dowex 50W-X8(H⁺ form). The decarboxylase was periodically added to the mixture (each0.2 mL) at an interval of 30 min, to avoid the denaturation which iscaused by the rapid formation of acetaldehyde. The total amount of theenzyme as 3.2 mL (200 U). The reaction mixture was further stirredovernight. Then the mixture was centrifuged, and the supernatant wasdiluted to 100 mL and applied to a column of Dowex 1-X8 (20-50 mesh,bicarbonate form, bed volume, 100 mL). The pH of the eluent and washingswas re-adjusted to 5.5 and further applied to the same column to ensurethe adsorption of desired product. After washing with water, the desiredproduct was eluted with a linear gradient from 0 to 0.3M of ammoniumbicarbonate. The product was further purified by Biogel P-2 column (bedvolume 20 mL) to give 192 mg (33%) of 18. The ¹ H NMR spectrum wasidentical with the sample mentioned above.

Example 12 Benzyl2,4,5,7,8-penta-O-acetyl-3-deoxy-α-D-manno-2-octulosonate (20b)

A suspension of KDO ammonium salt monohydrate (160 mg, 0.59 mol), aceticanhydride (3 mL), pyridine (3 mL), and 4-(N,N-dimethylamino)pyridine(DMAP, 2 mg) was stirred overnight at room temperature. Ice-cooled waterwas added and the mixture was stirred for 30 min. After dilution withwater, the pH of the mixture was adjusted to 3.5 by addition of Dowex50W-X8 (H⁺ form). The resin was filtered off, and the filtrate wasconcentrated in vacuo. The residue was diluted with a mixture ofchloroform and toluene and the solvent was evaporated. This procedurewas repeated three times to remove trace of water. The residue wasdissolved in anhydrous DMF. Benzyl bromide (161 mg, 0.94 mmol), Cs₂ CO₃(390 mg, 1.20 mmol), and tetrabutylammonium iodide (33 mg) were addedand the mixture was stirred for 4 h at room temperature under N₂. Themixture was diluted with 0.5N ice-cooled hydrochloric acid and extractedtwice with a mixture of diethyl ether and toluene (1:1). The organiclayer was successively washed with water, saturated aqueous NaHCO₃ andbrine, dried over anhydrous Na₂ SO₄ and concentrated in vacuo. Theresidue was chromatographed over silica gel (20 g). Elution withhexane-diethyl ether (2:1-1:1) afforded 15b, which was recrystallizedfrom diethyl ether to give 220 mg (70%) as colorless plates, mp102°-103° C. (lit.^(26b) mp 98°-99° C.); [α]²⁶ D +293° (c 1.0, CHCl₃)[lit.^(26b) [α]²⁵ D +91.9° (c 0.9, CHCl₃). Its ¹ H NMR spectrum (CDCl₃)was in good accordance with that reported previously by Nakamoto (Chem.Pharm. Bull. 1987, 35, 4537). HRMS (M+Na⁺) calcd 561.1584, found561.1602.

Example 13 2,4,5,6,8-Penta-O-acetyl-3-deoxy-α-D-manno-2-octulosonic acid(20a)

A mixture of 20b (220 mg, 0.41 mmol) and Pd-C (10%, 55 mg) in ethanol(3mL) was vigorously stirred under H₂ at room temperature for 1 h. Afterthe catalyst was filtered off, the filtrate was concentrated in vacuo.The residue was recrystallized from diethyl ether to give 20a (177 mg,97%) as fine needles, mp 132°-133° C.; [α]²⁵ D +374° (c 0.88, CHCl₃).Its ¹ H NMR spectrum (C₆ D₆) was identical with that reported previouslyby Unger et al. (Carbohydr. Res. 1980, 80, 191).

Example 14 1,3,4,6,7-Penta-O-acetyl-2-deoxy-β-D-manno-heptose (21)

To a solution of acid chloride prepared from 20a (30 mg, 0.067 mmol) intoluene was added dropwise a solution of N-hydroxythiopyridone 22 (11mg,0.09 mmol) and DMAP (2 mg) in toluene (0.5 mL) and pyridine (0.3 mL) atroom temperature under N₂ in the dark. After stirring for 10 min,t-butylmercaptane (0.5 mL) was added and the mixture was irradiated withwhite light (tungsten lamp, 100 W) at room temperature. After stirringfor 10 min, N₂ was introduced to the mixture under a slightly reducedpressure to remove residual t-butylmercaptane for 30 min. Usual workupand purification by silica gel preparative TLC [developed withhexane-Et₂ O (1:1)] afforded 21 (18.5 mg, 68% as an oil, [α]²² D +36.8°(c 1.85, CHCl₃); ¹ H NMR (CDCl₃) δ 2.000-2.150 (2H, m, H-2ax, H-2eq),2.010 (6H, s, acetyl), 2.082 (3H, s, acetyl), 2.119 (3H, s, acetyl),2.137 (3H, s, acetyl), 3.882 (1H, dd, J₅,4 =1.5, J₅,6 =10.0 Hz, H-5),4.115 (1H, dd, J_(7'),6 =4.5, J_(7'),7 =12.5 H, H-7'), 4.437 (1H, dd,J₇,6 =2.5, J₇,7' =12.5 Hz, H-7), 5.073 (1H, ddd, J₃,4 =3.0, J₃.2eq =5.0,J₃,2ax =12.5 Hz, H-3), 5.165 (1H, ddd, J₆,7 =2.5, J₆,7' =4.5, J₆,5 =10.0Hz, H-6), 5.303 (1H, dd, J₄,5 =1.5, J₄,3 =3.0 Hz, H-4), 5.748 (1H, dd,J₁,2eq =3.0, J₁,2ax =10.0 Hz, H-1); ¹³ C NMR (CDCl₃) δ 20.59, 20.59,20.65, 20.65, 20.84, 30.35, 62.26, 63.84, 67.32, 67.90, 71.62, 91.67,168.60, 169.60, 169.83, 170.30, 170.54. HRMS (M+Cs⁺) calcd C₁₇ H₂₄ O₁₁Cs 537.0373, found 537.0359.

Example 154-Acetamido-1,3,6,7,8-Penta-O-acetyl-2,4-dideoxy-α-D-glycero-D-galacto-octose(24)

A 25 mL two-necked flask equipped with septum, micro-scale Dean-Starktrapp which was filled with molecular sieves 4A, and a reflux condenser,was used as the reaction vessel. A mixture of 23a (35.0 mg, 0.70 mmol),DMAP (12.3 mg, 1.5 eq), 22 (41.0 mg, 5.0 eq), triethylamine (19 μL) inCH₂ Cl₂ (1 mL) was placed in the flask as above. To this wassuccessively added a solution of WSCI-Cl (20 mg) in CH₂ Cl₂ (1 mL) andt-butylmercaptane (0.5 mL). The mixture was stirred and irradiated withwhite light (tungsten lamp, 100 W) at room temperature for 5 h. Thereaction was worked up in a similar manner as described above. The crudeproduce was purified by silica gel preparative TLC [developed with ethylacetate-tetrahydrofuran (1:1)] to give 24 (8.7 mg, 27% from 23a) as anoil, [α]²² D +21.3° (c 2.87, CHCl₃); ¹ H NMR (CDCl₃) δ 1.908 (3H, s,N-acetyl), 1.915 (1H, ddd, J_(2ax),1 =10.3, J_(2ax),3 =11.5, J_(2ax),2eq=12.4 Hz, H-2ax), 2.043 (3H, s, O-acetyl), 2.051 (3H, s, O-acetyl),2.102 (3H, s, O-acetyl), 2.107 (3H, s, O-acetyl), 2.134 (3H, s,O-acetyl), 2.219 (1H, ddd, J_(2eq),1 =2.1, J_(2eq),3 =4.9, J_(2eq),2ax=12.4 Hz, H-2eq), 3.764 (1H, dd, J₅,6 =2.4, J₅,4 =10.4 Hz, H-5), 4.023(1H, dd, J₈,7 =5.5, J₈,8' =12.6 Hz, H-8), 4.062 (1H, ddd, J₄,NH =10.0,J₄,3 =10.3, J₄,5 =10.4 Hz, H-4), 4.389 (1H, dd, J_(8'7) =2.6, J_(8'),8=12.6 Hz, H-8'), 5.127 (1H, ddd, J₇,8' =2.6, J₇,8 =5.5, J₇,6 =7.3 Hz,H-7), 5.058 (1H, ddd, J₃,2eq =4.9, J₃,4 =10.3, J₃,2ax =11.5 Hz, H-3),5.190 (1H, d, J_(NH),4 =10.0 Hz, NH), 5.391 (1H, dd, J₆,7 =7.3, J₆,5=2.4 Hz, H-6), 5.646 (1H, dd, J₁,2eq =2.1, J₁,2ax =10.3 Hz, H-1); ¹³ CNMR (CDCl₃) δ 20.70, 20.70, 20.75, 20.83, 20.83, 23.15, 35.09, 49.22,61.98, 67.11, 70.23, 70.23, 73.67, 91.19, 168.75, 169.90, 170.12,170.36, 170.59, 170.88. HRMS (M+Cs⁺) calcd C₂₀ H₂₉ O₁₂ NCs 608.0744,found 608.0750.

What is claimed is:
 1. A purified 2-keto-3-deoxy-octulosonic acid (KDO)aldolase for catalytically condensing an aldose with pyruvate forforming 2-keto-deoxy-onic acid wherein the aldose is selected from thegroup consisting of D-ribose, D-threose, D-erythrose, 2-deoxy-D-ribose,L-glyceraldehyde, D-glyceraldehyde, 2-deoxy-2-fluoro-D-arabinose,D-lyxose, 5-azido-2,5-dideoxy-D-ribose, D-altrose, L-mannose, andanalogs thereof, said purified KDO aldolase being characterized asfollows:the KDO aldolase having a first substrate specificity withrespect to catalytically condensing D-arabinose with pyruvate forforming 3-deoxy-D-manno-2-octulsonic acid and a second substratespecificity with respect to catalytically condensing D-ribose withpyruvate for forming 3-deoxy-α-D-altro-2-octulosonic, wherein the secondsubstrate specificity with respect to D-ribose is at least 7% of thefirst substrate specificity with respect to D-arabinose; and saidpurified KDO aldolase is isolated from Aureobacterium barkerei strainKDO-37-2 (ATCC Accession Number 49977).
 2. The purified KDO aldolase asdescribed in claim 1 wherein:K_(m) for the KDO aldolase with respect tothe condensation of D-arabinose is approximately 1.2M at a pH ofapproximately 7.5 and a temperature of approximately 37° C.
 3. Thepurified KDO aldolase as described in claim 2 wherein:V_(max) for theKDO aldolase with respect to the condensation of D-arabinose isapproximately 0.73 U/mg at a pH of approximately 7.5 and a temperatureof approximately 37° C.